Robustness of archaeal populations in anaerobic co-digestion of dairy and poultry wastes

The objective of this study is to investigate the responses of methanogen populations to poultry waste addition by comparing the archaeal microbial populations in continuous anaerobic digesters with or without the addition of poultry waste as a co-substrate. Poultry waste was characterized as an organic/nitrogen-rich substrate for anaerobic digestion. Supplementing dilute dairy waste with poultry waste for anaerobic co-digestion to increase organic loading rate by 50% resulted in improved biogas production. Elevated ammonia derived from poultry waste did not lead to process inhibition at the organic loadings tested, demonstrating the feasibility of the anaerobic co-digestion of dairy and poultry wastes for improved treatment efficiency. The stability of the anaerobic co-digestion process was linked to the robust archaeal microbial community, which remained mostly unchanged in community structure following increases in organic loading and ammonia levels. Surprisingly, Crenarchaeota archaeal populations, instead of the Euryarchaeota methanogens, dominated the archaeal communities in the anaerobic digesters. The ecological functions of these abundant non-methanogen archaeal populations in anaerobic digestion remain to be identified.     

Effects of heat treatment on hydrogen production potential and microbial community of thermophilic compost enrichment cultures

Cellulosic plant and waste materials are potential resources for fermentative hydrogen production. In this study, hydrogen producing, cellulolytic cultures were enriched from compost material at 52, 60 and 70 °C. Highest cellulose degradation and highest H₂ yield were 57% and 1.4 mol-H₂ mol-hexose⁻¹ (2.4 mol-H₂ mol-hexose-degraded⁻¹), respectively, obtained at 52 °C with the heat-treated (80 °C for 20 min) enrichment culture. Heat-treatments as well as the sequential enrichments decreased the diversity of microbial communities. The enrichments contained mainly bacteria from families Thermoanaerobacteriaceae and Clostridiaceae, from which a bacterium closely related to Thermoanaerobium thermosaccharolyticum was mainly responsible for hydrogen production and bacteria closely related to Clostridium cellulosi and Clostridium stercorarium were responsible for cellulose degradation.     

Functional similarities and differences of an archaeal Hsp70(DnaK) stress protein compared with its homologue from the bacterium Escherichia coli

Archaea are prokaryotes but some of their chaperoning systems resemble those of eukaryotes. Also, not all archaea possess the stress protein Hsp70(DnaK), in contrast with bacteria and eukaryotes, which possess it without any known exception. Further, the primary structure of the archaeal DnaK resembles more the bacterial than the eukaryotic homologues. The work reported here addresses two questions: Is the archaeal Hsp70 protein a chaperone, like its homologues in the other two phylogenetic domains? And, if so, is the chaperoning mechanism of bacterial or eukaryotic type? The data have shown that the DnaK protein of the archaeon Methanosarcina mazei functions efficiently as a chaperone in luciferase renaturation in vitro, and that it requires DnaJ, and the other bacterial-type chaperone, GrpE, to perform its function. The M. mazei DnaK chaperone activity was enhanced by interaction with the bacterial co-chaperone DnaJ, but not by the eukaryotic homologue HDJ-2. Both the bacterial GrpE and DnaJ stimulated the ATPase activity of the M. mazei DnaK. The M. mazei DnaK-dependent chaperoning pathway in vitro is similar to that of the bacterium Escherichia coli used for comparison. However, in vivo analyses indicate that there are also significant differences. The M. mazei dnaJ and grpE genes rescued E.coli mutants lacking these genes, but E.coli dnaK mutants were not complemented by the M. mazei dnaK gene. Thus, while the data from in vitro tests demonstrate functional similarities between the M. mazei and E.coli DnaK proteins, in vivo results indicate that, intracellularly, the chaperones from the two species differ.     

Chemical composition and antimicrobial activity of the essential oils of the Amazon Guatteriopsis species

The essential oils of Guatteriopsis blepharophylla, Guatteriopsis friesiana and Guatteriopsis hispida were obtained by hydrodistillation and analysed by GC and GC/MS. The main compound found in the leaf oil of G. blepharophylla was caryophyllene oxide (1) (69.25%). The leaf oil of G. friesiana contained predominantly beta-eudesmol (2) (51.60%), gamma-eudesmol (3) (23.70%), and alpha-eudesmol (4) (14.56%). The major constituents identified in the leaf of G. hispida were beta-pinene (38.18%), alpha-pinene (30.77%) and (E)-caryophyllene (20.59%). The antimicrobial activity of the essential oils was evaluated against 11 species of microorganisms. The oil of G. friesiana exhibited significant antimicrobial activity for all microorganisms tested, whereas that of G. hispida and G. blepharophyla had potent activity against Rhodococcus equi with MIC of 50 microg mL(-1). The major constituents of each oil were also tested separately, and showed lower activity compared to the oils. Moreover, mixtures of the main constituents, in the same proportions found in G. friesiana and G. hispida oils, did not show the same activity as the original oils.     

Probing the archaeal diversity of a mixed thermophilic bioleaching culture by TGGE and FISH

The archaeal community present in a sample of Mixed Thermophilic Culture-B (MTC-B) from a laboratory-scale thermophilic bioleaching reactor was investigated by temperature gradient gel electrophoresis (TGGE) and fluorescence in situ hybridisation (FISH). Both techniques were specifically adapted for use on native state bioleaching samples, with a view to establishing convenient means for monitoring culture composition. Using the TGGE protocol developed, the relative species composition of the thermophilic bioleaching sample was analysed, and included four phylotypes belonging to the Sulfolobales, which were related to Stygiolobus azoricus, Metallosphaera sp. J1, Acidianus infernus and Sulfurisphaera ohwakuensis. However, the St. azoricus-like phylotype was difficult to resolve and some micro-heterogeneity was observed within this phylotype. Specific FISH probes were designed to qualitatively assess the presence of the phylotypes in MTC-B. The sample was dominated by Sf. ohwakuensis-like Archaea. In addition, the St. azoricus-like, Metallosphaera species-like and Acidianus species-like cells appeared in similar low abundance in the community. Most strikingly, FISH identified Sulfolobus shibatae-like cells present in low numbers in the sample even though these were not detected by PCR-dependent TGGE. These results highlight the importance of using more than one molecular technique when investigating the archaeal diversity of complex bioleaching reactor samples.     

An experimental test of the effects of variation in recruitment intensity on intertidal community composition

The rocky intertidal has been a model system for experimentally testing hypotheses regarding the factors that structure natural communities. Many ecologists have proposed that changes in the intensity of recruitment of individuals into a community should influence community structure. Past work investigating this hypothesis has primarily been surveys of recruitment and community structure across large spatial scales. Surprisingly, no researchers to date have manipulated recruitment into a rocky intertidal community to assess the response of interactions of the whole community to variation in recruitment intensity. We manipulated the densities of Balanus glandula and Chthamalus spp. recruits across a four-fold difference at two sites spanning the Monterey Bay, California, USA, to experimentally test if differences in recruitment intensity influenced initial changes in community composition and if these changes persisted through time. The results of this work indicate that differences in recruitment influence community composition initially, but that these changes can be short-lived.     

An unusual ring—A opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila

A series of steroids (progesterone, testosterone acetate, 17β-acetoxy-5α-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17β side-chain.This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17β-acetoxy-5α-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17β-alcohol of testosterone.All steroidal metabolites were isolated by column chromatography and were identified by ¹H, ¹³C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.     

Enhancement of transglycosylation activity by construction of chimeras between mesophilic and thermophilic beta-glucosidase

The family 3 beta-glucosidase from Thermotoga maritima is a highly thermostable enzyme (85 degrees C) that displays transglycosylation activity. In contrast, the beta-glucosidase from Cellvibrio gilvus is mesophilic (35 degrees C) and displays no such transglycosylation activity. Both enzymes consist of two domains, an N-terminal and a C-terminal domain, and the amino acid identities between the two enzymes in these domains are 32.4 and 36.4%, respectively. In an attempt to identify the molecular basis underpinning the display of transglycosylation activity and the requirements for thermal stability, eight chimeric genes were constructed by shuffling the two parental beta-glucosidase genes at four selected borders, two in the N-terminal domain and two in the C-terminal domain. Of the eight chimeric genes constructed, only two chimeric enzymes (Tm578/606Cg and Tm638/666Cg) gave catalytically active forms and these were the ones shuffled in the C-terminal domain. For these active chimeric enzymes, 80% (Tm578/606Cg) and 88% (Tm638/666Cg) of their amino acid sequences originated from T. maritima. With regard to their thermal profiles, the two active chimeric enzymes, Tm578/606Cg and Tm638/666Cg, displayed profiles intermediate to those of the two parental enzymes as they were optimally active at 65 and 70 degrees C, respectively. These two chimeric enzymes were optimally active at pH 4.1 and 3.9, which is closer to that observed for the T. maritima enzyme (pH 3.2-3.5) than that for the C. gilvus enzyme (pH 6.2-6.5). Kinetic parameters for the chimeric enzymes were investigated with five different substrates including pNP-beta-D-glucopyranoside. The kinetic parameters obtained for the chimeric enzymes were closer to those of the T. maritima enzyme than to those of the C. gilvus enzyme. Transglycosylation activity was observed for both chimeric enzymes and the activity of the Tm578/606Cg chimera was at a level twice that observed with the T. maritima enzyme. This study is an effective demonstration of the usefulness of chimeric enzymes in altering the characteristics of an enzyme.     

The effect of a tube-building phoronid on associated infaunal species diversity, composition and community structure

Habitat modifying organisms can alter the distribution of associated species. We surveyed soft-sediment patches in Bodega Harbor, California and found that patches with high densities of the phoronid Phoronopsis harmeri (Pixell, 1912), a chemically-defended tube-building lophophorate, have higher infaunal abundance and richness than similar patches with low densities of P. harmeri. To determine whether this difference was driven by P. harmeri and whether this difference is attributable to the activities of the organism, or simply its physical structure, we conducted a field experiment with four treatments: live phoronids, mimics of phoronid structure, phoronid-free sediments (bare) and unmanipulated sediments. Although the field experiment did not detect differences in the overall abundance or richness of infauna among the manipulated treatments, some of the individual species did show a positive response to the presence of phoronids and phoronid structure (i.e., mimics). In particular, the polychaete Boccardia proboscidea, the amphipod Monocorophium uenoi, and harpacticoid copepods were facilitated by the presence of phoronids and phoronid structure when there was sediment disturbance. The inconsistency between the results of the survey and of the manipulative experiment may be largely driven by the disturbance caused by the manipulation. However, where P. harmeri has an effect, it is generally positively associated with infaunal abundance that may be attributable to the stabilization of sediments.     

Insecticidal activity and chemical composition of the essential oil of Artemisia vestita from China against Sitophilus zeamais

In our screening program for new agrochemicals from local wild plants, essential oil of Artemisia vestita Wall (Asteraceae) was found to possess strong insecticidal activity against maize weevil, Sitophilus zeamais Motsch. Essential oil of aerial parts of A. vestita was obtained from hydrodistillation and was investigated by GC and GC–MS. The main components of essential oil were grandisol (40.29%), 1,8-cineol (14.88%) and camphor (11.37%). The essential oil of A. vestita possessed strong fumigant toxicity against S. zeamais adults with a LC₅₀ value of 13.42 mg/L air. The essential oil of A. vestita also showed contact toxicity against S. zeamais adults with a LD₅₀ value of 50.62 mg/adult.     

Chemical composition and antimicrobial activity of the essential oil of Scutellaria barbata

The essential oil of Scutellaria barbata was obtained by hydrodistillation with a 0.3% (v/w) yield and analysed by GC and GC-MS. The main compounds in the oil were hexahydrofarnesylacetone (11.0%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (7.8%), menthol (7.7%) and 1-octen-3-ol (7.1%). The antimicrobial activity of the oil was evaluated against 17 microorganisms using disc diffusion and broth microdilution methods. The gram-positive bacteria, including methicillin-resistant Staphlococcus aureus, were more sensitive to the oil than gram-negative bacteria and yeasts.     

Transcapsidation and the conserved interactions of two major structural proteins of a pair of phytoreoviruses confirm the mechanism of assembly of the outer capsid layer

The strongly conserved amino acid sequences of the P8 outer capsid proteins of Rice dwarf virus (RDV) and Rice gall dwarf virus (RGDV) and the distribution of electrostatic potential on the proteins at the interfaces between structural proteins suggested the possibility that P8-trimers of RGDV might bind to the 3-fold symmetrical axes of RDV core particles, with vertical interaction between heterologous P3 and P8 proteins and lateral binding of homologous P8 proteins, thereby allowing formation of the double-layered capsids that are characteristic of viruses that belong to the family Reoviridae. We proved this hypothesis using chimeric virus-like particles composed of the P3 core capsid protein of RDV and the P8 outer capsid protein of RGDV, which were co-expressed in a baculovirus expression system. This is the first report on the molecular biological proof of the mechanism of the assembly of the double-layered capsids with disparate icosahedral lattices.     

Effect of fungal infection on the reproductive potential of aphids and their progeny

The effect of infection by Pandora neoaphidis and Beauveria bassiana on the reproductive potential of the pea aphid, Acyrthosiphon pisum, and their progeny was assessed. Infection by either P. neoaphidis or B. bassiana reduced the number of nymphs produced within 24 h of inoculation and over the entire infection period compared to uninfected aphids. However, infection by either P. neoaphidis or B. bassiana for 24 or 72 h did not alter the intrinsic rate of increase of the host aphid's progeny. Therefore, fungal infection appears to have no indirect effects on the fitness of the host's progeny.     

Influence of organic co-solvents on the activity and substrate specificity of feruloyl esterases

Organic co-solvents can expand the use of enzymes in lignocellulose deconstruction through making substrates more soluble and thus more accessible. In choosing the most adequate co-solvent for feruloyl esterases, hydrolysis of methyl p-hydroxycinnamates by three pure enzymes (and a multi-enzyme preparation) was evaluated. Low concentrations of dimethylsulfoxide (DMSO) enhanced hydrolysis by two of the enzymes while at levels >20%, activity was reduced. DMSO also enhanced acetyl esterase-type activity of the enzymes. The co-solvent effect was different for each enzyme-substrate couple, indicating that other factors are also involved. Kinetic studies with a Talaromyces stipitatus feruloyl esterase showed low concentrations of dimethylsulfoxide enhanced the hydrolytic rate while K_m also increased. Moreover, long-term incubation (96 h) of an Aspergillus niger feruloyl esterase in dimethylsulfoxide:water provided to the enzyme the ability to hydrolyze methyl p-coumarate, suggesting an active-site re-arrangement. Dimethylsulfoxide (10-30%) is proposed as an adequate co-solvent for feruloyl esterase treatment of water-insoluble substrates.     

Comparison of static,in-vessel composting of MSW with thermophilic anaerobic digestion and combinations of the two processes

The biological stabilisation of the organic fraction of municipal solid waste (OFMSW) into a form stable enough for land application can be achieved via aerobic or anaerobic treatments. To investigate the rates of degradation (e.g. via electron equivalents removed, or via carbon emitted) of aerobic and anaerobic treatment, OFMSW samples were exposed to computer controlled laboratory-scale aerobic (static in-vessel composting), and anaerobic (thermophilic anaerobic digestion with liquor recycle) treatment individually and in combination. A comparison of the degradation rates, based on electron flow revealed that provided a suitable inoculum was used, anaerobic digestion was the faster of the two waste conversion process. In addition to faster maximum substrate oxidation rates, anaerobic digestion (followed by post-treatment aerobic maturation), when compared to static composting alone, converted a larger fraction of the organics to gaseous end-products (CO₂ and CH₄), leading to improved end-product stability and maturity, as measured by compost self-heating and root elongation tests, respectively. While not comparable to windrow and other mixed, highly aerated compost systems, our results show that in the thermophilic, in-vessel treatment investigated here, the inclusion of a anaerobic phase, rather than using composting alone, improved hydrolysis rates as well as oxidation rates and product stability. The combination of the two methods, as used in the DiCOM^® process, was also tested allowing heat generation to thermophilic operating temperature, biogas recovery and a low odour stable end-product within 19 days of operation.     

In vivo photosystem I reduction in thermophilic and mesophilic cyanobacteria: the thermal resistance of the process is limited by factors other than the unfolding of the partners

Photosystem I reduction by plastocyanin and cytochrome c(6) in cyanobacteria has been extensively studied in vitro, but much less information is provided on this process inside the cell. Here, we report an analysis of the electron transfer from both plastocyanin and cytochrome c(6) to photosystem I in intact cells of several cyanobacterial species, including a comparative study of the temperature effect in mesophilic and thermophilic organisms. Our data show that cytochrome c(6) reduces photosystem I by following a reaction mechanism involving complex formation, whereas the copper-protein follows a simpler collisional mechanism. These results contrast with previous kinetic studies in vitro. The effect of temperature on photosystem I reduction leads us to conclude that the thermal resistance of this process is determined by factors other than the proper stability of the protein partners.     

A novel endo-glucanase from the thermophilic bacterium Geobacillus sp. 70PC53 with high activity and stability over a broad range of temperatures

A thermophilic Geobacillus bacterium secreting high activity of endo-glucanase (EC 3.2.1.4) was isolated from rice straw compost supplemented with pig manure. A full-length gene of 1,104 bp, celA, encoding this glycosyl hydrolase family 5 endo-glucanase of 368 amino acids was isolated. No related gene from Geobacillus has been reported previously. The recombinant CelA expressed in Escherichia coli had an optimal activity at 65°C and pH 5.0, and it exhibited tenfold greater specific activity than the commercially available Trichoderma reesei endo-glucanase. CelA displayed activity over a broad temperature range from 45 to 75°C and was a thermostable enzyme with 90% activity retained after heating at 65°C for 6 h. Interestingly, CelA activity could be enhanced by 100% in the presence of 2 mM MnSO₄. CelA had high specific activity over β-d-glucan from barley and Lichenan, making it a potentially useful enzyme in biofuel and food industries.     

Temporal and spatial windows delimit activation of the outer ring of wingless in the Drosophila wing

Extracellular signalling molecules play many roles in the development of higher organisms. They are used reiteratively in different tissues and stages, but the response of the receiving cells is controlled in a context dependent manner. The pattern of expression of the signalling molecule Wingless/WNT in Drosophila is extraordinarily complex. We have studied the mechanism that controls its expression and function in the outer ring of the Drosophila wing hinge. Our findings indicate that wingless expression is controlled by a dual mechanism: its initial activation requires the product of zinc finger homeodomain 2 and is subsequently repressed by the product of the gene complex elbow/no ocelli. This tight regulation restricts the activation of wingless temporally and spatially. Later in development, wingless expression is maintained by an autoregulatory loop that involves the product of homothorax. We have analyzed the phenotype of a wingless allelic combination that specifically removes the outer ring, and our results show that Wingless is required to promote local proliferation of the wing base cells. Thus, cell proliferation in the proximal-distal axis is controlled by the sequential activation of wingless in the inner ring and the outer ring at different stages of development.     

Egg production of the copepod Acartia bifilosa in two contrasting European estuaries in relation to seston composition

The egg production of the copepod Acartia bifilosa was measured and related to environmental variables and food availability in two estuaries located in the same biogeographic region (Bay of Biscay) but showing very strong differences in abiotic and biotic features: the Gironde estuary (France) and the estuary of Mundaka (Spain). The study was conducted during the spring-summer-autumn period of 1994. Food availability was evaluated by analysing the chlorophyll a (Chl a), the particulate organic carbon (POC) and the easily extractable macromolecular compounds such as proteins, carbohydrates and lipids of the seston. The egg production of copepods was estimated from field incubations with natural water, and phytoplankton feeding of adult females was estimated by means of the gut fluorescence method. The nutritional environment of the Gironde was characterised by high amounts of suspended particulate matter (SPM) with low food value, emphasising the mainly detrital origin of the organic matter (OM). In Mundaka, the higher contribution of phytoplankton to the seston led to marked increases in particulate food value accounting for up to 35% of organic matter. The weight-specific egg production was found to be sharply higher in Mundaka (ranging from 0.2 to 0.63×10⁻³ day⁻¹) than in the Gironde (ranging from 0 to 0.13×10⁻³ day⁻¹), but the seasonal trend of variations was similar, the highest weight-specific egg production rates occurring in early summer and the lowest in autumn in both estuaries. Egg production was not correlated linearly with temperature since maximal egg production occurred at intermediate temperatures. In Mundaka, the egg production showed a significant positive correlation with the chlorophyll and the Chl/SPM and the POC/SPM ratios. This coupled with higher values of algal food availability (Chl a/SPM: 10 to 1870 μg g⁻¹) and gut fluorescence (between 0.12 and 0.38 ng Chl a Eq ind⁻¹) indicate that a herbivorous diet could cover the energy requirements of A. bifilosa and support egg production. In the Gironde, the algal food availability and the gut fluorescence were lower (Chl a/SPM: 10 to 80 μg g⁻¹; GF: 0.09 and 0.25 ng Chl a Eq ind⁻¹), and the egg production showed significant positive correlation with the particulate food value, suggesting that other sources of carbon rather than phytoplankton were responsible for the observed changes in egg production. Results indicate that the particular seston properties of each system may be responsible for the noticeable differences in A. bifilosa fertility among estuaries.